Transcriptional transactivation by selected short random peptides attached to lexA-GFP fusion proteins

Abstract

Background: Transcriptional transactivation is a process with remarkable tolerance for sequence diversity and structural geometry. In studies of the features that constitute transactivating functions, acidity has remained one of the most common characteristics observed among native activation domains and activator peptides.

Results: We performed a deliberate search of random peptide libraries for peptides capable of conferring transcriptional transactivation on the lexA DNA binding domain. Two libraries, one composed of C-terminal fusions, the other of peptide insertions within the green fluorescent protein structure, were used. We show that (i) peptide sequences other than C-terminal fusions can confer transactivation; (ii) though acidic activator peptides are more common, charge neutral and basic peptides can function as activators; and (iii) peptides as short as 11 amino acids behave in a modular fashion.

Conclusions: These results support the recruitment model of transcriptional activation and, combined with other studies, suggest the possibility of using activator peptides in a variety of applications, including drug development work.

Figure 1

Cartoon of peptide libraries fused to lexA molecules. +GPF is a truncated GFP molecule of 157 amino acids. CAS is the 11-residue core activating sequence (see Results).

Figure 2

Graph of net charge of lexA-GFP/peptide activator sequences. Charge is calculated assuming that D = E = -1, K = R = +1. Ordinate is the fraction of sequences with a given charge.

Figure 3

Graph of net charge of lexA-peptide activator sequences (as in Figure 2).

Figure 4

Bargraph of sequence features displayed by random library clones vs. activator sequences.

Figure 5

Bar graph showing results of colony formation assay. 250 yeast cells were plated on leu+ plates and leu-plates and grown at 30°C for two days. The fraction of cells that formed colonies on a leu-plate compared to a leu+ plate is plotted.

Figure 6

Comparison between native activation domain and activator peptide. Patches of yeast cells on a selection (leu-) plate.